title: Essential glycan-dependent interactions optimize MHC class I peptide loading.
abstract: In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the peptide-loading complex (PLC). We provide evidence for an MHC class I/CRT intermediate before PLC engagement and examine the nature of that chaperone interaction in detail. To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC subcomplex, excluding the Transporter Associated with Antigen Processing, from purified, recombinant proteins. ERp57 disulfide linked to the class I-specific chaperone tapasin and CRT were the minimal PLC components required for MHC class I association and peptide loading. Mutations disrupting the interaction of CRT with ERp57 or the class I glycan completely eliminated PLC activity in vitro. By using the purified system, we also provide direct evidence for a role for UDP-glucose:glycoprotein glucosyltransferase 1 in MHC class I assembly. The recombinant Drosophila enzyme reglucosylated MHC class I molecules associated with suboptimal ligands and allowed PLC reengagement and high-affinity peptide exchange. Collectively, the data indicate that CRT in the PLC enhances weak tapasin/class I interactions in a manner that is glycan-dependent and regulated by UDP-glucose:glycoprotein glucosyltransferase 1.
authors.affiliation: Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8011, USA.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Wearsch
authors.forename: David R
authors.initials: DR
authors.lastname: Peaper
authors.forename: Peter
authors.initials: P
authors.lastname: Cresswell
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology*; D037282:Calreticulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004331:Drosophila melanogaster; D005964:Glucosyltransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011134:Polysaccharides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D037282:Calreticulin; D015395:Histocompatibility Antigens Class I; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D011134:Polysaccharides; D011994:Recombinant Proteins; C107762:tapasin; D005964:Glucosyltransferases; C489364:glucosyltransferase I; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human